Microfluidic in-line labeling method for continuous-flow protease inhibition analysis

• US 6,468,761 B2
• Filed: 01/05/2001
• Issued: 10/22/2002
• Est. Priority Date: 01/07/2000
• Status: Active Grant

• Current Assignee: Caliper Holdings Incorporated
• Original Assignee: Caliper Holdings Incorporated
• Inventors: Yang, Hua, Sundberg, Steven
• Primary Examiner(s): Leary, Louise N.
• Application Number:
  US09/755,608
• Publication Number:
  US 20010026929A1
• Time in Patent Office: 655 Days
• Field of Search: 435/23, 435/24, 435/283.1, 435/287.1, 435/286.1, 435/286.5, 435/4
• US Class Current: 435/23
• CPC Class Codes: B01L 2200/10 Integrating sample preparation and analysis in single entity, e.g. lab-on-a-chip concept
• CPC Class Codes: B01L 2300/0816 Cards, e.g. flat sample carriers usually with flow in two horizontal directions
• CPC Class Codes: B01L 2300/0867 Multiple inlets and one sample wells, e.g. mixing, dilution
• CPC Class Codes: B01L 2400/0406 capillary forces
• CPC Class Codes: B01L 2400/0415 electrical forces, e.g. electrokinetic
• CPC Class Codes: B01L 2400/0487 fluid pressure, pneumatics
• CPC Class Codes: B01L 3/5027 by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip B01L3/5023 takes precedence; micromixers B01F33/30; microreactors for synthesis B01J19/0093; microcapillary devices in general B81B1/00
• CPC Class Codes: C12Q 1/37 involving peptidase or proteinase
• CPC Class Codes: G01N 33/6809 involving fluorescent derivatizing reagents reacting non-specifically with all amino acids

Abstract

Enzyme assays are performed in microfluidic devices including, e.g., in-line labeling, separation, and detection of assay products. In-line labeling allows assays, e.g., protease assays, to be performed in a continuous flow microfluidic format. Also included are microfluidic devices and integrated systems for performing in-line labeling in continuous flow enzyme assays.

Claims

1. A method of performing a protease assay in a microfluidic system, the method comprising:

(i) flowing a protease through at least one microscale cavity;

(ii) contacting the protease with a protease substrate in the at least one microscale cavity or an additional microscale cavity, thereby reacting the protease substrate with the protease and producing one or more products;

(iii) labeling the one or more products by flowing a labeling reagent into the at least one microscale cavity or into the additional microscale cavity to react with the one or more products and produce one or more labeled products.

2. The method of claim 1, wherein the one or more products comprise at least one unlabeled amino acid, peptide, or protein.

3. The method of claim 1, wherein the labeling reagent comprises a chemically reactive labeling reagent.

4. The method of claim 1, wherein the labeling reagent comprises an amine-derivatizing reagent.

5. The method of claim 4, wherein the amine-derivatizing reagent comprises a non-fluorescent reagent prior to reacting the labeling reagent with the one or more products.

6. The method of claim 1, wherein the labeling reagent comprises a heterocyclic aromatic carbamate compound.

7. The method of claim 6, wherein the heterocyclic aromatic carbamate compound is 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate.

8. The method of claim 6, wherein the heterocyclic aromatic carbamate compound is selected from:

3-aminoquinolyl-N-hydroxysuccinimidyl carbamate, 5-aminoquinolyl-N-hydroxysuccinimidyl carbamate, 5-aminoisoquinolyl-N-hydroxysuccinimidyl carbamate, 6-amino-4-methylquinolyl-N-hydroxysuccinimidyl carbamate, 6-amino-2,4-dimethylquinolyl-N-hydroxysuccinimidylcarbamate, 6-amino-2phenylquinolyl-N-hydroxysuccinimidyl-carbamate, 6-amino-2-methoxy-4-methylquinolyl-N-hydroxysuccinimidylcarbamate, 4-aminoquinaldine-N-hydroxysuccinimidyl carbamate, 9-aminoacridine-N-hydroxysuccinimidyl carbamate, 2-aminoacridine-N-hydroxysuccinimidylcarbamate, luminol-N-hydroxysuccinimidylcarbamate, isoluminol-N-hydroxysuccinimidylcarbamate, 7-amino4-methylcoumarin-N-hydroxysuccinimidylcarbamate, 7-amino-4-(trifluoromethyl)coumarin-N-hydroxysuccinimidylcarbamate, 4′

-(aminomethyl)fluorescein-N-hydroxysuccinimidylcarbamate, 5-(aminomethyl)fluorescein-N-hydroxysuccinimidylcarbamate, 5-aminoeosin-N-hydroxysuccinimidylcarbamate, and Cascade Blue ethylenediamine-N-hydroxysuccinimidylcarbamate.

9. The method of claim 4, wherein the labeling reagent comprises 3-(4-carboxybenzoylquinoline-2-carboxaldehyde, 3-(2-furosyl) quinoline-2-carboxaldehyde, fluorescamine, or 7-nitrobenz-2-oxa-1,3-diazole chloride.

10. The method of claim 4, wherein the labeling reagent comprises an aromatic dialdehyde.

11. The method of claim 10, wherein the aromatic dialdehyde is selected from:

o-pthaldialdehyde, napthalene-2-3-dicarboxaldehyde, and anthracene-2,3-dicarboxaldehyde.

12. The method of claim 1, wherein the labeling step comprises chemically reacting the labeling reagent with the one or more products.

13. The method of claim 1, comprising flowing one or more of:

the protease, the protease substrate, the one or more products, the one or more labeled products, and the labeling reagent, through the microfluidic system by applying pressure or electrokinetic forces to one or more of;

the protease, the protease substrate, the one or more products, the one or more labeled products, and the labeling reagent, in the microfluidic system.

14. The method of claim 1, further comprising separating the one or more products or the one or more labeled products in the microscale cavity or in the additional microscale cavity.

15. The method of claim 14, wherein the separating comprises electrophoretically separating the one or more products or the one or more labeled products in a polymer, a gel, or a solution.

16. The method of claim 15, wherein the polymer, gel, or solution comprises polyacrylamide, linear polyacrylamide, cross-linked polyacrylamide, polydimethylacrylamide, polydimethylacrylamide/co-acrylic acid, agarose or cellulose.

17. The method of claim 1, further comprising flowing an inactivating reagent through the at least one microscale cavity or the additional microscale cavity after the labeling step, thereby inactivating any labeling reagents that have not reacted with the one or more products.

18. The method of claim 17, wherein flowing an inactivating reagent through the at least one microscale cavity or the additional microscale cavity comprises altering the pH of the enzyme, the substrate, the products, or the labeled products.

19. The method of claim 1, further comprising contacting the enzyme and substrate with one or more modulators.

20. The method of claim 19, wherein the one or more modulators comprise at least one inhibitor or at least one activator.

21. The method of claim 1, further comprising providing a detection system and detecting the one or more labeled products with the detection system.

22. The method of claim 21, wherein detecting comprises fluorescently detecting the one or more labeled products.

23. The method of claim 21, further comprising providing a computer, operably coupled to the detection system, the computer comprising software, which software comprises at least a first instruction set, which first instruction set quantitates the amount of the one or more labeled products detected by the detection system.

24. A method of labeling one or more products in a microfluidic system, the method comprising:

(i) flowing one or more unlabeled components through at least one microfluidic cavity;

(ii) reacting the one or more unlabeled components with one or more unlabeled reagents in the at least one microscale cavity or in an additional microscale cavity, resulting in one or more unlabeled products; and

,

(iii) labeling the one or more unlabeled products by flowing a heterocyclic aromatic carbamate compound into contact with the one or more unlabeled products in the at least one microscale cavity or in the additional microscale cavity, thereby reacting the heterocyclic aromatic carbamate compound with the one or more unlabeled products and producing one or more labeled products.

25. A microfluidic device for in-line labeling, the device comprising a body structure having a plurality of microscale channels disposed therein, the plurality of channels comprising:

(i) a main channel;

in which main channel at least one protease and at least one protease substrate are reacted to produce one or more unlabeled products;

(ii) a labeling channel region fluidly coupled to the main channel;

in which labeling channel region the one or more unlabeled products are labeled to produce one or more labeled products;

(iii) a detection channel region fluidly coupled to the main channel, in which detection channel region the one or more labeled products are detected;

(iv) at least a first source fluidly coupled to the main channel, which first source comprises an amine-derivatizing reagent, which reagent reacts with the one or more unlabeled products to produce the one or more labeled products.

26. The device of claim 25, wherein at least one of the one or more unlabeled products comprises an unlabeled protein, a peptide, or an amino acid.

27. The device of claim 25, wherein the amine-derivatizing reagent is a heterocyclic aromatic carbamate compound.

28. The device of claim 27, wherein the heterocyclic aromatic carbamate compound comprises 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate.

29. The device of claim 27, wherein the heterocyclic aromatic carbamate compound is selected from:

3-aminoquinolyl-N-hydroxysuccinimidyl carbamate, 5-aminoquinolyl-N-hydroxysuccinimidyl carbamate, 5-aminoisoquinolyl-N-hydroxysuccinimidyl carbamate, 6-amino-4-methylquinolyl-N-hydroxysuccinimidyl carbamate, 6-amino-2,4-dimethylquinolyl-N-hydroxysuccinimidylcarbamate, 6-amino-2phenylquinolyl-N-hydroxysuccinimidyl-carbamate, 6-amino-2-methoxy-4-methylquinolyl-N-hydroxysuccinimidylcarbamate, 4-aminoquinaldine-N-hydroxysuccinimidyl carbamate, 9-aminoacridine-N-hydroxysuccinimidyl carbamate, 2-aminoacridine-N-hydroxysuccinimidylcarbamate, luminol-N-hydroxysuccinimidylcarbamate, isoluminol-N-hydroxysuccinimidylcarbamate, 7-amino4-methylcoumarin-N-hydroxysuccinimidylcarbamate, 7-amino-4-(trifluoromethyl)coumarin-N-hydroxysuccinimidylcarbamate, 4′

-(aminomethyl)fluorescein-N-hydroxysuccinimidylcarbamate, 5-(aminomethyl)fluorescein-N-hydroxysuccinimidylcarbamate, 5-aminoeosin-N-hydroxysuccinimidylcarbamate, and Cascade Blue ethylenediamine-N-hydroxysuccinimidylcarbamate.

30. The device of claim 25, wherein the amine-derivatizing reagent comprises 3-(4-carboxybenzoylquinoline-2-carboxaldehyde, 3-(2-furosyl) quinoline-2-carboxaldehyde, fluorescamine, or 7-nitrobenz-2-oxa-1,3-diazole chloride.

31. The device of claim 25, wherein the amine-derivatizing reagent comprises an aromatic dialdehyde.

32. The device of claim 31, wherein the aromatic dialdehyde is selected from:

o-pthaldialdehyde, napthalene-2-3-dicarboxaldehyde, and anthracene-2,3-dicarboxaldehyde.

33. The device of claim 25, wherein the device further comprises at least a second source, which second source comprises a protease.

34. The device of claim 25, wherein the device further comprises at least a second source, which second source comprises a protease substrate.

35. The device of claim 25, wherein the device further comprises a second source, which second source comprises an inactivating reagent.

36. The device of claim 35, wherein the inactivating reagent alters the pH of one or more of:

the protease, the protease substrate, the unlabeled products, and the labeled products.

37. The device of claim 25, the device further comprising a separation channel region for separating the one or more unlabeled products or the one or more labeled products, which separation channel region is fluidly coupled to the main channel.

38. The device of claim 37, wherein the separation channel region comprises a polymer, gel, or solution.

39. The device of claim 38, wherein the polymer, gel, or solution comprises polyacrylamide or polydimethylacrylamide/co-acrylic acid.

40. A system for performing in-line labeling in a microfluidic device, the system comprising:

(i) a microfluidic device comprising a plurality of microscale channels disposed therein, the plurality of microscale channels comprising;

(a) a main channel;

in which main channel at least one protease and at least one protease substrate are reacted to produce one or more unlabeled products;

(b) a labeling channel region fluidly coupled to the main channel;

in which labeling channel region the one or more unlabeled products are labeled to produce one or more labeled products;

(c) a detection channel region fluidly coupled to the main channel, in which detection channel region the one or more labeled products are detected;

(d) at least a first source fluidly coupled to the main channel, which first source comprises an amine-derivatizing reagent, which reagent reacts with the one or more unlabeled products to produce the one or more labeled products;

(ii) a fluid direction system fluidly coupled to the microfluidic device, which fluid direction system transports one or more of;

the at least one protease, the at least one protease substrate, the one or more unlabeled products, the one or more labeled products, and the amine-derivatizing reagent, through the plurality of microscale channels;

(iii) a control system operably linked to the fluid direction system, which control system instructs the fluid direction system to transport one or more of;

the at least one protease, the at least one protease substrate, the one or more unlabeled products, the one or more labeled products, and the amine-derivatizing reagent, through the plurality of microscale channels; and

,

(iv) a detection system positioned proximal to the detection channel region for detecting the one or more labeled products.

41. The system of claim 40, wherein the fluid direction system transports one or more of:

the at least one protease, the at least one protease substrate, the one or more unlabeled products, the one or more labeled products, and the amine-derivatizing reagent, through the plurality of microscale channels by applying pressure or electrokinetic forces to one or more of;

the at least one protease, the at least one protease substrate, the one or more unlabeled products, the one or more labeled products, and the amine-derivatizing reagent.

42. The system of claim 40, the system further comprising a computer operably coupled to the system, and software, which software comprises at least a first instruction set.

43. The system of claim 42, wherein the first instruction set analyzes signals produced from the detection system.

44. The system of claim 42, wherein the first instruction set quantitates signals produced from the detection system.

45. The system of claim 42, wherein the first instruction set directs fluid movement in the system.

46. The system of claim 45, wherein the first instruction set directs the at least one protease and the at least one protease substrate to flow through the main channel;

directs the amine-derivatizing reagent to flow into contact with the one or more unlabeled products in the labeling channel region, thereby producing labeled products; and

directs the movement of the labeled products through the detection channel region.